Riboswitches are non-coding messenger RNA (mRNA) elements that bind metabolites with high specificity to mediate gene expression control (Roth 2009). Riboswitches of numerous forms and functions, which recognize chemically diverse ligands with high selectivity, have been identified in bacteria, plants and fungi. The ubiquity of such mRNA regulatory elements highlights the need for a deeper understanding of their mechanism and mode of action.
As riboswitch domains are believed to regulate numerous essential genes in bacterial organisms, methods and reagents enabling direct insights into the riboswitch controlled transcription and translation regulation may have significant value in the discovery and/or tailoring of therapeutic agents specifically targeting one or more riboswitch domains.
Bacterial riboswitches generally operate through cis-acting control mechanisms, where a specific metabolite binds an aptamer domain in the mRNA's 5′-untranslated region in a manner that influences its propensity to adopt a specific fold (Roth 2009; Montange 2008). Structural investigations suggest that riboswitches respond to a metabolite's presence by adopting ‘on’ or ‘off’ conformations of the expression platform domain that up- or down-regulate the transcriptional or translational fate of the genes in which they are found (Blouin 2009). The capacity to influence transcriptional or translational expression in a ligand-dependent manner stipulates that a riboswitch adopts distinct ligand-free and ligand-bound conformations that exchange on a time scale that is timed with respect to the regulated event. The time scale of such dynamics and the nature of riboswitch conformational flexibility ensure a that the default regulatory pathway is favored in the absence of ligand. These events are also tuned to enable adequate time for metabolite recognition and for the conformational transition responsible for the regulatory outcome. A rapid response could be achieved if the riboswitch spontaneously and transiently adopts ligand-binding competent conformations. However, to avoid spurious signaling, such states must be unstable to ensure that the default folding pathway predominates in the absence of ligand.
Thus, methods and reagents that enable the direct assessment of riboswitch dynamics offer a unique means of assessing the regulatory switch underpinning riboswitch function and for evaluating responsiveness to specific metabolites and/or ligands.
Little is known about the nature of the apo-riboswitch conformation and how ligand-sensing transduces conformational changes in the expression platform to achieve alternative regulatory outcomes. (Garst 2009; Stoddard 2010; Baird 2010b; Edwards 2010)
To address these open questions, we present a detailed investigation of the ligand-induced folding process of the S-adenosylmethionine type II (SAM-II) riboswitch, as a model system for assessing riboswitch function since it possesses a compact fold, where both the aptamer domain and the expression platform domain (also referred to as the regulatory switch domain) are contiguous and defined by just 50 nucleotides. S-adenosylmethionine (SAM) is an important cofactor that represents the key methyl group donor for the methylation of various biomolecules. In bacteria, SAM is involved in methionine biosynthesis and related sulfur metabolic pathways. Its synthesis is frequently regulated via a riboswitch mechanism. Previous studies describe a total of five classes of SAM-binding riboswitches that each contains a binding pocket capable of discriminating among near-cognate derivatives, e.g., S-adenosylhomocysteine (SAH) (Wang 2008; Poiata 2009). The SAM-II riboswitch, occurring predominantly in proteobacteria, represent the class which is smallest in size (Corbino 2005). The crystal structure of a SAM-II riboswitch (from the 5′-UTR of the metX gene discovered in the Sargasso Sea metagenome) bound to SAM has been determined (Gilbert 2008). The findings revealed that the SAM-II riboswitch folds into a classical H-type pseudoknot (FIG. 1), where Loop L1 interacts with the major groove of the P2b helix in a triple helical arrangement. The Shine-Dalgarno (SD) sequence ( . . . AAAG50G51G52-3′), a determinant of the expression platform domain of this riboswitch, lies within the 3′-purine-rich domain of the complex. This nucleoside stretch becomes masked by the riboswitch fold forming numerous hydrogen bonding contacts along the major groove of the P2b helix. The terminal residues of this region form stem P2a through two Watson-Crick base pairs with Loop L1 (G51:C15 and G50:C16).
In the course of the investigations leading to the present invention, the ligand-induced folding pathway of the SAM-II riboswitch was explored using a series of chemical and biophysical methods, including NMR, fluorescence spectroscopy and single-molecule fluorescence resonance energy transfer (smFRET) imaging (Haller 2011). The elucidated data indicated this riboswitch follows a multistep folding pathway that entails a dynamic conformational switching mechanism. Furthermore, the studies revealed that regional tertiary structure elements in the SAM-II riboswitch fold on distinct time scales, suggesting an ordered sequence of events in the ligand recognition process. These measurements shed light on the intrinsically dynamic nature of the SAM-II riboswitch, its propensity to exchange between distinct conformations and how these processes may impact the regulatory circuit of riboswitches in the regulatory control of gene expression.
Accordingly, the strategy, reagents and methods outlined in the present invention are directly applicable to other riboswitch domains of diverse types and functions. The direct implication from the model system employed is that riboswitch-mediated regulation hinges on metabolite and/or ligand-modulated dynamics, where ligand binding has a direct consequence on the mobility of the expression platform domain. Hence, the present invention addresses the need to follow structural changes of the expression platform domain under ligand binding and other conditions which was heretofore unavailable, and provides new reagents and methods for performing FRET analysis of the riboswitch.